This invention relates to the enumeration of cells in a cell sample, for example in a bone marrow cell sample, a body fluid sample, a disaggregated tissue sample, a tissue fine needle aspiration sample or, in particular, a blood sample. More particularly, the invention relates to a method suitable for, but not limited to, enumerating the number of CD4+ lymphocytes in a blood sample.
There are several methods for performing cell counting which are currently available. The most popular method is flow cytometric cell testing. Non-flow cytometric methods are also available, although these methods are less widely used.
Flow Cytometric CD4+ T Cell Testing
Two methods of flow cytometric CD4 testing are utilised worldwide for CD4+ T cell enumeration. The first method involves the use of two machines and is referred to as the dual or double platform method (DP) (1). This method involves the use of specific panels of antibodies including CD3, CD4, CD8, CD19, CD16, CD56, and CD45. Various fluorochrome combinations can be used to form either two, three or four colour combinations of antibody to constitute the various panels. Duplication of at least one measurement to ensure reproducibility e.g. CD3, is typically performed. In some instances it is recommended that CD14 expression is also used in the panel to exclude CD14 positive monocytes from the “lymphocyte” gate and ensure purity of the lymphocyte population studied.
The dual platform method makes use of a haematology analyser to obtain an absolute lymphocyte count (ALC) [which is obtained by multiplication of two independently measured parameters viz. the white cell count and the % lymphocytes of the total white blood cell differential]. A flow cytometer is used to obtain a corresponding CD4 percentage (CD4%) of lymphocytes.
The CD4 antigen expressed on the surface of T cells and monocytes plays an important role in the MHC class II-restricted responses of specific T lymphocytes. It also serves as the major receptor of human immunodeficiency viruses (HIV).
CD45 is a trans-membrane protein tyrosine phosphatase (also called Leucocytee Common Antigen) that is expressed on all haematopoietic cells. Expression is at a higher density on lymphocytes whilst the expression is lower on other leukocytes like granulocytes and monocytes. In addition to the blood, bone marrow and lymphatics, since CD45 is found throughout the hematopoietic lineage, CD45 will be found wherever such cells constitute a significant portion of the tissue, e.g. spleen, thymus, and bone marrow.
Lymphocytes are defined by flow cytometry either through dual light scatter parameters, or alternatively (now considered the only recommended method in new guidelines) by the use of dual bright CD45 expression with side scatter properties. The lymphocyte population is used as the common denominator or reference point between the haematology and flow cytometry machines in order to calculate a CD4 T cell of total lymphocytes.
The absolute CD4 count is calculated by multiplying the CD4% (which can alternatively be defined as CD3+/CD4+ T cells) of the total lymphoid population defined by flow cytometry by the ALC, which is then expressed as number of cells per microlitre, or number of cells×106/l, or the number of cells×109/l.
Inter-laboratory variation ranging between 15-44% is, however, described for the widely used dual platform method (greater than 60% of returns in a recent U.K. NEQAS Immunology shipment have been reported). Theoretically, in order that all laboratories measure lymphocytes equally, the U.K. NEQAS, CDC, NCCLS, and others have devised very specific guidelines (1) for use of a “Lymphosum”. The “Lymphosum” itself is a method of identifying the total lymphoid population by flow cytometry including all T cells (CD3+), all B cells (CD19+) and all Natural Killer cells (CD16+/56+), so that the sum of the individual components equals approximately 100% of lymphocytes.
Various gating strategies that include identification of bright CD45 positive cells (lymphocytes) have been recommended to ensure purity of the lymphoid population (2) and improve the quality of the assay, but this is not widely practiced in many countries. The practice of abbreviated panels, including only CD3/CD4 and CD8 is based loosely on international guidelines. It is practiced largely due to cost containment, and has limited quality control, especially on samples which are older than 6-24 hours, when cell disintegration results in poor cellular definition and hence problems occur with accurate identification and subsequent gating of lymphocytes. Use of CD3, CD4 and CD8 antibodies in a single tube to determine the percentage of CD4 positive T cells is therefore not recommended for use (1) as there is no means in the abbreviated panel of CD3, CD4 and CD8 only to validate the purity or completeness of the lymphocyte gate.
From the haematology analyser perspective, white blood cell counts and especially automated differential counts should ideally be performed within 6 hours of venesection and preferably within 24 hours. There is currently no known haematology analyser manufactured that reliably facilitates white cell and/or white blood cell differential counting after 36 hours after blood collection.
Further, different haematology analysers use different methods of identifying lymphocytes and show variation in ALC reporting according to the specifications of the analyser, even on fresh samples less than 6 hours old. Although the guidelines for identifying lymphocytes by flow cytometry are relatively clear, to date no recommended or universal method of identifying lymphocytes or other different types of white blood cell on a haematology analyser exists, despite there being existing knowledge of very specific differential expression of CD45 on lymphocytes. ALC white blood cell differential counts are therefore neither quality controlled nor standardised between laboratories and results are therefore unreliable and not directly comparable between laboratories. Because the CD4 count is calculated from the ALC in the dual platform system, it follows that the inaccuracies of the ALC are translated to the CD4 count. Thus, the error in each independent step of each the separate systems in the dual platform method is multiplied at each subsequent step in the calculation.
In spite of the relatively wide inter-laboratory variation of CD4 result reporting, the dual platform flow cytometry system remains the most widely used and preferred system for CD4 testing. One of the main problems of the dual platform method lies in the identification of lymphocytes as the common denominator or reference point between the two platforms. An Absolute Lymphocyte Count (ALC) is a highly variable parameter between laboratories. Currently, although white blood cell (WBC) counts are well quality controlled, both internally on the instruments themselves and externally on various quality assessment schemes, white blood cell differential counting methods and techniques of identifying lymphocytes on various haematology analysers are neither quality controlled or standardised. As ALCs are currently used as the common denominator and mainstay of dual platform system CD4 counting, it therefore follows that the documented variability of ALC reporting is carried over into CD4+ T cell result reporting.
Single Platform Testing
The single platform method (SP) of measuring CD4+ T cells uses flow cytometry only, and both the cell counting and the identification of the CD4+ T cells are performed on the same instrument, a flow cytometer. It is recommended due to the improved reproducibility of laboratory results, the inter-laboratory variation having been reported as varying between 10-18%.
There are several options for performing single platform CD4 testing. One option includes the use of a flow cytometer with a volumetric precision counting facility, e.g. the Ortho CytoronAbsolute (Ortho Diagnostic Systems, USA) but this instrument is no longer manufactured. The second option utilises beads as a reference standard from which the cells themselves can be counted. The beads are added to the sample in a known concentration, either during manufacture or during sample preparation, in a specified volume, to a similar volume of blood and counted alongside the cells of interest on a flow cytometer. Bead-based counting, although less variable between laboratories, is, however, more technically intensive and relies heavily on accurate, precise pipetting, and also on the technical skills of the operator. Duplication of testing is also recommended to assess pipetting error and ensure accuracy of counting. This increases further the costs of bead-based testing.
Simplified, smaller single platform flow cytometers dedicated to CD4 enumeration (FACSCount, BDS, San Jose, Calif., USA) are also widely used. Although these instruments are considerably cheaper than flow cytometers, reagents costs for these instruments are more costly than for ordinary flow cytometry, thereby also prohibiting their use in many laboratories.
An additional problem of both the single platform and dual platform systems is that of the increased costs associated with “Lymphosum” testing. Unfortunately the obviously less expensive “CD4 only” alternatives do not offer sufficient built-in quality control to ensure accuracy and precision of individual sample testing.
Alternative Technologies for CD4+ T Cell Determination.
There are also several alternative non-flow cytometric technologies for CD4+ T cell counting available. These include the Dynabead™ assay, Coulter Cytospheres Assay TRAx assay (and Microvolume Fluorimetry The Absolute Lymphocyte Count (ALC) has also been proposed as an alternative to CD4 enumeration where an ALC of less than 1000 cells/μl is used a substitutes for a CD4+ T cell count of 200/μl or less. Poor correlation of these parameters, however, has been shown, and it has been suggested that an ALC substitute does not offer meaningful information for individual patient management.
The current dual methods of CD4 testing are therefore impractical and/or expensive, and do not provide results which are sufficiently accurate. A need thus exists to provide a method Which is simple to perform, accomodates existing technologies and is more accurate than currently methods employed.